Compositions and methods for detection of babesia

ABSTRACT

Methods for the rapid detection of the presence or absence of Babesia in a biological or non-biological sample are described. The methods can include performing an amplifying step, a hybridizing step, and a detecting step. Furthermore, primers and probes targeting Babesia and kits are provided that are designed for the detection of Babesia, including, but not limited to, the Babesia species of B. microti, B. divergens, B. duncani, and B. venatorum. Also described are kits, reaction mixtures, and oligonucleotides (e.g., primer and probe) for the amplification and detection of Babesia.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a Continuation patent application of U.S. patentapplication Ser. No. 16/031,320, filed Jul. 10, 2018, which claims thebenefit of priority of U.S. Provisional Patent Application No.62/534,046, filed Jul. 18, 2017, both of which are incorporated hereinby reference in their entireties.

FIELD OF THE INVENTION

The present disclosure relates to the field of in vitro diagnostics.Within this field, the present invention concerns the amplification anddetection of a target nucleic acid that may be present in a sample andparticularly, the amplification, detection, and quantitation of a targetnucleic acid comprising sequence variations and/or individual mutationsof Babesia species, using primers and probes. The invention furtherprovides reaction mixtures and kits containing primers and probes foramplification and detection of Babesia.

BACKGROUND OF THE INVENTION

Babesia (also known as Nuttallia) is a protozoan parasite that infectsred blood cells, causing a disease known as babesiosis (also known aspiroplasmosis). Babesia is usually tick-borne (and tick-transmitted) butis also transmissible by transfusion or from mother to child duringpregnancy or delivery.

Most cases of babesiosis are asymptomatic and symptoms, if they occur,are non-specific and may include flu-like symptoms (i.e., fever, chills,sweats, headache, myalgia, and arthralgia), hemolytic anemia, orthrombocytopenia. Babesiosis is particularly life threatening inpatients with asplenia, weakened immune systems (e.g., due to cancerlymphoma, or AIDS), co-morbodities, such as liver or kidney disease, orwho are over the age of 50. In such patients, multi-organ dysfunction,disseminated intravascular coagulation, and even death, may occur(Vannier, et al., Infect. Dis. Clin. N. Am. 29:357-370 (2015)). Babesiaparasites reproduce in red blood cells, where they are seen ascross-shaped inclusions and cause hemolytic anemia, not unlike malaria.Due to historical misclassifications, babesiosis has also been known asTexas cattle fever, redwater fever, tick fever, and Nantucket fever.Babesiosis is a malaria-like parasitic disease and is regarded as thesecond-most common blood parasite of mammals, and has a major impact onthe health of domestic animals and humans. Although human babesiosis hashistorically been uncommon, it is an emerging disease in theNortheastern and Midwestern United States as well as parts of Europe.

Babesia is considered to be the second most commonly found bloodparasite of mammals (after trypanosomes). More than one hundred speciesof Babesia have been identified. The vast majority oftransfusion-associated cases of Babesia infection in the U.S. are due tothe species Babesia microti, and roughly 2% of reported cases are due toBabesia duncani. Tick-transmission of B. microti mainly occurs in sevenstates in the Northeast (Connecticut, Maine, Massachusetts, NewHampshire, New Jersey, New York, and Rhode Island) and the upper Midwest(particularly Minnesota and Wisconsin) of the U.S. (see, Herwaldt, etal., Annals of Internal Medicine 155(8):509-520 (2011)). B. duncani, isendemic in the West Coast of the U.S. (see, Herwaldt, et al. (2011)). In2011, there were 1,124 cases of babesiosis reported in the U.S., ofwhich 10 cases were transfusion-associated (see, Center for DiseaseControl and Prevention: Morbidity and Mortality Weekly Report61(27):501-515 (2012)). From 1979-2009, a period spanning 30 years, 162cases of transfusion-associated babesiosis were reported, at a rateincreasing over time (see, Herwaldt, et al. (2011)). Notwithstandingthat these statistics may significantly underestimate the true rate oftransfusion-associated babesiosis, Babesia is the most commonlytransmitted transfusion-associated infection (Leiby, Annals of InternalMedicine 155(8):556-557 (2011)). To date, no Babesia test for screeningblood donors has been licensed, and no pathogen-reduction technologiesfor Babesia are available in the U.S. Although a history of babesiosisinfection is a ground for indefinite deferral as a blood donor, manydonors may not be aware that they carry the parasite and may haveasymptomatic parasitemia and remain infectious for over a year. Further,the Babesia parasite is viable in blood products. The majority oftransfusion-associated cases are associated with erythrocytes (includingleukoreduced or irradiated units), with a few cases due to platelettransfusion. Prospective testing of blood donations in endemic areas ofthe U.S. has yielded a 0.38% positive rate for Babesia (Moritz, et al.,N. Engl. J. Med. 375(23):2236-2245 (2016)).

Because no FDA-licensed screening tests are available, to date, the U.S.blood supply is not currently screened for Babesia. Accordingly,clinicians may miss the diagnosis of transfusion-associated babesiosis,because the clinical presentation is non-specific, and the nationwidedistribution of blood products means that cases can occur outside ofareas of high Babesia prevalence and outside of peak summer months oftick-borne disease.

Although there are methods (i.e., immunological methods) for detectingBabesia in blood and other tissue and/or biological samples, thesemethods lack sensitivity and do not accurately predict infectivity ofBabesia in blood. Moreover, such immunological methods cannot detectinfection during the period when Babesia is present, but has notelicited enough antibodies sufficient for detection. Like otherinfectious diseases for which blood donations are screened, blooddonations must be screened with a sensitive assay to detect Babesia sothat infected units may be interdicted and discarded.

In the field of molecular diagnostics, the amplification and detectionof nucleic acids is of considerable significance. Such methods can beemployed to detect any number of microorganisms, such as viruses andbacteria. The most prominent and widely-used amplification technique isthe Polymerase Chain Reaction (PCR). Other amplification techniquesinclude Ligase Chain Reaction, Polymerase Ligase Chain Reaction,Gap-LCR, Repair Chain Reaction, 3 SR, NASBA, Strand DisplacementAmplification (SDA), Transcription Mediated Amplification (TMA), andQβ-amplification. Automated systems for PCR-based analysis often makeuse of a real-time detection of product amplification during the PCRprocess in the same reaction vessel. Key to such methods is the use ofmodified oligonucleotides carrying reporter groups or labels.

Thus, rates of Babesia infection are dramatically increasing within theU.S., with no reliable sensitive assay or means for its detection insamples. Absent a reliably sensitive means for detecting Babesia, theincrease in Babesia infection rates, in particular, threatens the safetyof the blood donor supply. Therefore, there is a need in the art for aquick, reliable, specific, and sensitive method for detecting andquantifying the presence of Babesia in a sample.

SUMMARY OF THE INVENTION

Certain embodiments in the present disclosure relate to methods for therapid detection of the presence or absence of Babesia in a biological ornon-biological sample, for example, multiplex detection and quantitatingof Babesia by real-time polymerase chain reaction (PCR) in a single testtube or vessel. Embodiments include methods of detection of Babesiacomprising performing at least one cycling step, which may include anamplifying step and a hybridizing step. Furthermore, embodiments includeprimers, probes, and kits that are designed for the detection of Babesiain a single tube or vessel.

One embodiment of the invention is directed to a method of detectingBabesia in a sample, the method comprising: (a) performing anamplification step comprising contacting the sample with one or more setof primers to produce an amplification product, if a target nucleic acidof Babesia is present in the sample; (b) performing a hybridization stepcomprising contacting the amplification product, if the target nucleicacid of Babesia is present in the sample, with one or more probes; and(c) detecting the presence or absence of the amplification product,wherein the presence of the amplification product is indicative of thepresence of Babesia in the sample, and wherein the absence of theamplification product is indicative of the absence of Babesia in thesample; and wherein the one or more set of primers comprise one or moreprimers comprising a nucleic sequence of any one of a group consistingof SEQ ID NOs:1, 3, 4, 6, and 7, or a complement thereof; and whereinthe one or more probes comprise a nucleic acid sequence of any one of agroup consisting of SEQ ID NOs:2 and 5, or a complement thereof. In oneembodiment, the Babesia comprises any one or more Babesia species of agroup consisting of B. microti, B. divergens, B. duncani, and B.venatorum. In other embodiment, the Babesia species consists of theBabesia species B. microti. In one embodiment, the one or more set ofprimers comprise a first primer comprising a nucleic acid sequence ofSEQ ID NO:1, or a complement thereof, and a second primer comprising anucleic acid sequence of SEQ ID NO:3, or a complement thereof; andwherein the one or more probes comprise a nucleic acid sequence of SEQID NO:2, or a complement thereof. In another embodiment, the Babesiaspecies consists of any one or more of the Babesia species B. divergens,B. duncani, and B. venatorum. In a related embodiment, the one or moreset of primers comprise a primer comprising a nucleic acid sequence ofSEQ ID NO:4, or a complement thereof, and one or more primers comprisinga nucleic acid sequence of any one or more of a group consisting of SEQID NOs:6 and 7, or a complement thereof; and wherein the one or moreprobes comprise a nucleic acid sequence of SEQ ID NO:5, or a complementthereof. In one embodiment, the one or more second primers comprising anucleic acid sequence of any one or more of a group consisting of SEQ IDNOs:6 and 7, or a complement thereof, comprise a mixture of primers,wherein the mixture of primers comprises: (i) a primer comprising thenucleic acid sequence of SEQ ID NO:6, or a complement thereof, and (ii)a primer comprising the nucleic acid sequence of SEQ ID NO:7, or acomplement thereof. In another embodiment, the mixture of primerscomprises a mixture with equal amounts of: (i) a primer comprising thenucleic acid sequence of SEQ ID NO:6, or a complement thereof, and (ii)a primer comprising the nucleic acid sequence of SEQ ID NO:7, or acomplement thereof. In another embodiment, the Babesia species consistsof all of the following Babesia species: B. microti, B. divergens, B.duncani, and B. venatorum. In a related embodiment, the method ofdetecting B. microti comprises a set of primers for amplification of B.microti, and a probe for hybridizing to the amplification product of B.microti; and wherein the method of detecting B. divergens, B. duncani,and B. venatorum comprises a set of primers for amplification of B.divergens, B. duncani, and B. venatorum, and a probe for hybridizationto the amplification products of B. divergens, B. duncani, and B.venatorum. In a related embodiment, (a) the set of primers foramplification of B. microti comprises: (i) a first primer comprising thenucleic acid sequence of SEQ ID NO:1, or a complement thereof and (ii) asecond primer comprising the nucleic acid sequence of SEQ ID NO:3, or acomplement thereof; and the probe for hybridizing to the amplificationproduct of B. microti comprises the nucleic acid sequence of SEQ IDNO:2, or a complement thereof and (b) the set of primers foramplification of B. divergens, B. duncani, and B. venatorum comprises:(i) a primer comprising the nucleic acid sequence of SEQ ID NO:4, or acomplement thereof and (ii) one or more primers comprising a nucleicacid sequence of any one of a group consisting of SEQ ID NOs:6 and 7, ora complement thereof; and the probe for hybridizing to the amplificationproducts of B. divergens, B. duncani, and B. venatorum comprises thenucleic acid sequence of SEQ ID NO:5, or a complement thereof. Inanother embodiment, the one or more primers comprising a nucleic acidsequence of any one of a group consisting of SEQ ID NOs:6 and 7, or acomplement thereof, comprise a mixture of primers, wherein the mixtureof primers comprises: (i) a primer comprising the nucleic acid sequenceof SEQ ID NO:6, or a complement thereof, and (ii) a primer comprisingthe nucleic acid sequence of SEQ ID NO:7, or a complement thereof. In arelated embodiment, the mixture of primers comprises a mixture withequal amounts of: (i) a primer comprising the nucleic acid sequence ofSEQ ID NO:6, or a complement thereof, and (ii) a primer comprising thenucleic acid sequence of SEQ ID NO:7, or a complement thereof. Inanother embodiment, the sample is a biological sample, such as wholeblood, respiratory specimens, urine, fecal specimens, blood specimens,plasma, dermal swabs, nasal swabs, wound swabs, blood cultures, skin, orsoft tissue infections. In a related embodiment, the biological sampleis whole blood. In another embodiment, the hybridization step comprisescontacting the amplification product with the one or more probes,wherein the one or more probes is labeled with a donor fluorescentmoiety and a corresponding acceptor moiety; and the detecting stepcomprises detecting the presence or absence of fluorescent resonanceenergy transfer (FRET) between the donor fluorescent moiety and theacceptor moiety of the one or more probes, wherein the presence orabsence of fluorescence is indicative of the presence or absence ofBabesia in the sample. In a related embodiment, the donor fluorescentmoiety is HEX or FAM. In another embodiment, the acceptor moiety is aquencher, such as BlackHole Quencher™-2 (BHQ-2). In another embodiment,the donor fluorescent moiety and the acceptor moiety are within 5 to 20nucleotides of each other. In another embodiment, the donor fluorescentmoiety and the acceptor moiety are within 7 to 10 nucleotides of eachother. In another embodiment, the donor fluorescent moiety and theacceptor moiety are within 8 nucleotides of each other. In anotherembodiment, the donor fluorescent moiety and the acceptor moiety arewithin 10 nucleotides of each other.

Another embodiment of the invention is directed to a method of detectingBabesia in a sample, wherein the sample is whole blood, the methodcomprising: (a) performing an amplification step comprising contactingthe sample with one or more set of primers to produce an amplificationproduct, if a target nucleic acid of Babesia is present in the wholeblood sample; (b) performing a hybridization step comprising contactingthe amplification product, if the target nucleic acid of Babesia ispresent in the sample, with one or more probes; and (c) detecting thepresence or absence of the amplification product, wherein the presenceof the amplification product is indicative of the presence of Babesia inthe sample, and wherein the absence of the amplification product isindicative of the absence of Babesia in the sample; and wherein the oneor more set of primers comprises one or more primers comprising anucleic acid sequence of any one of a group consisting of SEQ ID NOs:1,3, 4, 6, and 7, or a complement thereof; and wherein the one or moreprobes comprise a nucleic acid sequence of any one of a group consistingof SEQ ID NOs:2 and 5, or a complement thereof. In another embodiment,the Babesia species comprises any one or more Babesia species of a groupconsisting of B. microti, B. divergens, B. duncani, and B. venatorum. Inanother embodiment, the method of detecting B. microti comprises a setof primers for amplification of B. microti, and a probe for hybridizingto the amplification product of B. microti; and wherein the method ofdetecting B. divergens, B. duncani, and B. venatorum comprises a set ofprimers for amplification of B. divergens, B. duncani, and B. venatorum,and a probe for hybridization to the amplification products of B.divergens, B. duncani, and B. venatorum. In another embodiment, (a) theset of primers for amplification of B. microti comprises: (i) a firstprimer comprising the nucleic acid sequence of SEQ ID NO:1, or acomplement thereof and (ii) a second primer comprising the nucleic acidsequence of SEQ ID NO:3, or a complement thereof; and the probe forhybridizing to the amplification product of B. microti comprises thenucleic acid sequence of SEQ ID NO:2, or a complement thereof; and (b)the set of primers for amplification of B. divergens, B. duncani, and B.venatorum comprises: (i) a primer comprising the nucleic acid sequenceof SEQ ID NO:4, or a complement thereof and (ii) one or more primerscomprising a nucleic acid sequence of any one of a group consisting ofSEQ ID NOs:6 and 7, or a complement thereof; and the probe forhybridizing to the amplification products of B. divergens, B. duncani,and B. venatorum comprises the nucleic acid sequence of SEQ ID NO:5, ora complement thereof. In another embodiment, the one or more primerscomprising a nucleic acid sequence of any one or more of a groupconsisting of SEQ ID NOs:6 and 7, or a complement thereof, comprises amixture of primers, wherein the mixture of primers comprises: (i) aprimer comprising the nucleic acid sequence of SEQ ID NO:6, or acomplement thereof, and (ii) a primer comprising the nucleic acidsequence of SEQ ID NO:7, or a complement thereof. In another embodiment,the mixture of primers comprises a mixture with equal amounts of: (i) aprimer comprising the nucleic acid sequence of SEQ ID NO:6, or acomplement thereof, and (ii) a primer comprising the nucleic acidsequence of SEQ ID NO:7, or a complement thereof.

Another embodiment of the invention is directed to a kit for detecting anucleic acid of Babesia that may be present in a sample, the kitcomprising amplification reagents comprising a DNA polymerase,nucleotide monomers, one or more set of primers comprising one or moreprimers comprising a nucleic acid sequence of any one of a groupconsisting of SEQ ID NOs:1, 3, 4, 6, and 7, or a complement thereof, andone or more probes comprising a nucleic acid sequence of any one of agroup consisting of SEQ ID NOs:2 and 5, or a complement thereof. Inanother embodiment, the Babesia comprises any one or more Babesiaspecies of a group consisting of B. microti, B. divergens, B. duncani,and B. venatorum. In another embodiment, the kit comprises a set ofprimers for amplification of B. microti, and a probe for hybridizing tothe amplification product of B. microti; and wherein the kit comprises aset of primers for amplification of B. divergens, B. duncani, and B.venatorum, and a probe for hybridization to the amplification productsof B. divergens, B. duncani, and B. venatorum. In another embodiment,(a) the set of primers for amplification of B. microti comprises: (i) afirst primer comprising the nucleic acid sequence of SEQ ID NO:1, or acomplement thereof and (ii) a second primer comprising the nucleic acidsequence of SEQ ID NO:3, or a complement thereof; and the probe forhybridizing to the amplification product of B. microti comprises thenucleic acid sequence of SEQ ID NO:2, or a complement thereof; and (b)the set of primers for amplification of B. divergens, B. duncani, and B.venatorum comprises: (i) a primer comprising the nucleic acid sequenceof SEQ ID NO:4, or a complement thereof; and (ii) one or more primerscomprising a nucleic acid sequence of any one of a group consisting ofSEQ ID NOs:6 and 7, or a complement thereof; and the probe forhybridizing to the amplification products of B. divergens, B. duncani,and B. venatorum comprises the nucleic acid sequence of SEQ ID NO:5, ora complement thereof. In another embodiment, the one or more primerscomprising a nucleic acid sequence of any one or more of a groupconsisting of SEQ ID NOs:6 and 7, or a complement thereof, comprises amixture of primers, wherein the mixture of primers comprises: (i) aprimer comprising the nucleic acid sequence of SEQ ID NO:6, or acomplement thereof, and (ii) a primer comprising the nucleic acidsequence of SEQ ID NO:7, or a complement thereof. In another embodiment,the mixture of primers comprises a mixture with equal amounts of: (i) aprimer comprising the nucleic acid sequence of SEQ ID NO:6, or acomplement thereof, and (ii) a primer comprising the nucleic acidsequence of SEQ ID NO:7, or a complement thereof. In another embodiment,the sample is a biological sample, such as whole blood, respiratoryspecimens, urine, fecal specimens, blood specimens, plasma, dermalswabs, nasal swabs, wound swabs, blood cultures, skin, or soft tissueinfections. In another embodiment, the one or more probes comprise adonor fluorescent moiety and a corresponding acceptor moiety. In arelated embodiment, the donor moiety is HEX or FAM. In anotherembodiment, the acceptor moiety is a quencher, such as BlackHoleQuencher™-2 (BHQ-2). In another embodiment, the donor fluorescent moietyand the acceptor moiety are within 5 to 20 nucleotides of each other. Inanother embodiment, the donor fluorescent moiety and the acceptor moietyare within 7 to 10 nucleotides of each other. In another embodiment, thedonor fluorescent moiety and the acceptor moiety are within 8nucleotides of each other. In another embodiment, the donor fluorescentmoiety and the acceptor moiety are within 10 nucleotides of each other.

Yet another embodiment of the invention is directed to one or more setof primers and one or more probes for the detection of Babesia in asample, wherein the one or more set of primers comprise one or moreprimers comprising a nucleic acid sequence of any one of a groupselected from SEQ ID NOs:1, 3, 4, 6, and 7, or a complement thereof; andwherein the one or more probes comprise a nucleic acid sequence of anyone of a group consisting of SEQ ID NOs:2 and 5, or a complementthereof. In another embodiment, the Babesia comprises any one or moreBabesia species of a group consisting of B. microti, B. divergens, B.duncani, and B. venatorum. In another embodiment, the one or more set ofprimers and one or more probes for detecting B. microti comprises a setof primers for amplification of B. microti, and a probe for hybridizingto the amplification product of B. microti; and wherein the one or moreset of primers and one or more probes for detecting B. divergens, B.duncani, and B. venatorum comprises a set of primers for amplificationof B. divergens, B. duncani, and B. venatorum, and a probe forhybridization to the amplification products of B. divergens, B. duncani,and B. venatorum. In another embodiment, (a) the set of primers foramplification of B. microti comprises: (i) a first primer comprising thenucleic acid sequence of SEQ ID NO:1, or a complement thereof; and (ii)a second primer comprising the nucleic acid sequence of SEQ ID NO:3, ora complement thereof; and the one or more probes for hybridizing to theamplification product of B. microti comprise the nucleic acid sequenceof SEQ ID NO:2, or a complement thereof; and (b) the set of primers foramplification of B. divergens, B. duncani, and B. venatorum comprises:(i) a primer comprising the nucleic acid sequence of SEQ ID NO:4, or acomplement thereof; and (ii) one or more primers comprising a nucleicacid sequence of any one of a group consisting of SEQ ID NOs:6 and 7, ora complement thereof; and the one or more probes for hybridizing to theamplification products of B. divergens, B. duncani, and B. venatorumcomprises the nucleic acid sequence of SEQ ID NO:5, or a complementthereof. In another embodiment, the one or more primers comprising anucleic acid sequence of any one or more of a group consisting of SEQ IDNOs:6 and 7, or a complement thereof, comprises a mixture of primers,wherein the mixture of primers comprises: (i) a primer comprising thenucleic acid sequence of SEQ ID NO:6, or a complement thereof, and (ii)a primer comprising the nucleic acid sequence of SEQ ID NO:7, or acomplement thereof. In another embodiment, the mixture of primerscomprises a mixture with equal amounts of: (i) a primer comprising thenucleic acid sequence of SEQ ID NO:6, or a complement thereof, and (ii)a primer comprising the nucleic acid sequence of SEQ ID NO:7, or acomplement thereof. In another embodiment, the one or more probescomprise a donor fluorescent moiety and a corresponding acceptor moiety.In a related embodiment, the donor moiety is HEX or FAM. In a anotherembodiment, the acceptor moiety is a quencher, such as BlackHoleQuencher™-2 (BHQ-2). In another embodiment, the donor fluorescent moietyand the acceptor moiety are within 5 to 20 nucleotides of each other. Inanother embodiment, the donor fluorescent moiety and the acceptor moietyare within 7 to 10 nucleotides of each other. In another embodiment, thedonor fluorescent moiety and the acceptor moiety are within 8nucleotides of each other. In another embodiment, the donor fluorescentmoiety and the acceptor moiety are within 10 nucleotides of each other.

Other embodiments provide an oligonucleotide comprising or consisting ofa sequence of nucleotides selected from SEQ ID NOs:1-7, or a complementthereof, which oligonucleotide has 100 or fewer nucleotides. In anotherembodiment, the present disclosure provides an oligonucleotide thatincludes a nucleic acid having at least 70% sequence identity (e.g., atleast 75%, 80%, 85%, 90% or 95%, etc.) to one of SEQ ID NOs:1-5, or acomplement thereof, which oligonucleotide has 100 or fewer nucleotides.Generally, these oligonucleotides may be primer nucleic acids, probenucleic acids, or the like in these embodiments. In certain of theseembodiments, the oligonucleotides have 40 or fewer nucleotides (e.g., 35or fewer nucleotides, 30 or fewer nucleotides, 25 or fewer nucleotides,20 or fewer nucleotides, 15 or fewer nucleotides, etc.) In someembodiments, the oligonucleotides comprise at least one modifiednucleotide, e.g., to alter nucleic acid hybridization stability relativeto unmodified nucleotides. Optionally, the oligonucleotides comprise atleast one label and optionally at least one quencher moiety. In someembodiments, the oligonucleotides include at least one conservativelymodified variation. “Conservatively modified variations” or, simply,“conservative variations” of a particular nucleic acid sequence refersto those nucleic acids, which encode identical or essentially identicalamino acid sequences, or, where the nucleic acid does not encode anamino acid sequence, to essentially identical sequences. One of skill inthe art will recognize that individual substitutions, deletions oradditions which alter, add or delete a single nucleotide or a smallpercentage of nucleotides (typically less than 5%, more typically lessthan 4%, 2% or 1%) in an encoded sequence are “conservatively modifiedvariations” where the alterations result in the deletion of an aminoacid, addition of an amino acid, or substitution of an amino acid with achemically similar amino acid.

In one aspect, amplification can employ a polymerase enzyme having 5′ to3′ nuclease activity. Thus, the donor fluorescent moiety and theacceptor moiety, e.g., a quencher, may be within no more than 5 to 20nucleotides (e.g., within 7 or 10 nucleotides) of each other along thelength of the probe. In another aspect, the probe includes a nucleicacid sequence that permits secondary structure formation. Such secondarystructure formation may result in spatial proximity between the firstand second fluorescent moiety. According to this method, the secondfluorescent moiety on the probe can be a quencher.

The present disclosure also provides for methods of detecting thepresence or absence of Babesia or Babesia nucleic acid, in a biologicalsample from an individual. These methods can be employed to detect thepresence or absence of Babesia nucleic acid in plasma, for use in bloodscreening and diagnostic testing. Additionally, the same test may beused by someone experienced in the art to assess urine and other sampletypes to detect and/or quantitate Babesia nucleic acid. Such methodsgenerally include performing at least one cycling step, which includesan amplifying step and a dye-binding step. Typically, the amplifyingstep includes contacting the sample with a plurality of pairs ofoligonucleotide primers to produce one or more amplification products ifa nucleic acid molecule is present in the sample, and the dye-bindingstep includes contacting the amplification product with adouble-stranded DNA binding dye. Such methods also include detecting thepresence or absence of binding of the double-stranded DNA binding dyeinto the amplification product, wherein the presence of binding isindicative of the presence of Babesia nucleic acid in the sample, andwherein the absence of binding is indicative of the absence of Babesianucleic acid in the sample. A representative double-stranded DNA bindingdye is ethidium bromide. Other nucleic acid-binding dyes include DAPI,Hoechst dyes, PicoGreen®, RiboGreen®, OliGreen®, and cyanine dyes suchas YO-YO® and SYBR® Green. In addition, such methods also can includedetermining the melting temperature between the amplification productand the double-stranded DNA binding dye, wherein the melting temperatureconfirms the presence or absence of Babesia nucleic acid nucleic acid.

In a further embodiment, a kit for detecting and/or quantitating one ormore nucleic acids of Babesia is provided. The kit can include one ormore sets of primers specific for amplification of the gene target; andone or more detectable oligonucleotide probes specific for detection ofthe amplification products.

In one aspect, the kit can include probes already labeled with donor andcorresponding acceptor moieties, e.g., another fluorescent moiety or adark quencher, or can include fluorophoric moieties for labeling theprobes. The kit can also include nucleoside triphosphates, nucleic acidpolymerase, and buffers necessary for the function of the nucleic acidpolymerase. The kit can also include a package insert and instructionsfor using the primers, probes, and fluorophoric moieties to detect thepresence or absence of Babesia nucleic acid in a sample.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although methods and materialssimilar or equivalent to those described herein can be used in thepractice or testing of the present subject matter, suitable methods andmaterials are described below. In addition, the materials, methods, andexamples are illustrative only and not intended to be limiting. Allpublications, patent applications, patents, and other referencesmentioned herein are incorporated by reference in their entirety. Incase of conflict, the present specification, including definitions, willcontrol.

The details of one or more embodiments of the invention are set forth inthe accompanying drawings and the description below. Other features,objects, and advantages of the invention will be apparent from thedrawings and detailed description, and from the claims.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows real time PCR growth curves of the B. microti nucleic acidtest (“395”) on pEF113 (B. microti) plasmid DNA dilutions (using primershaving a nucleic acid sequence of SEQ ID NOs:1 and 3 and a probe havinga nucleic acid sequence of SEQ ID NO:2).

FIG. 2 shows real time PCR growth curves of the B. microti nucleic acidtest (“395”) on B. microti genomic DNA (ATCC 30221D) dilutions (usingprimers having a nucleic acid sequence of SEQ ID NOs:1 and 3 and a probehaving a nucleic acid sequence of SEQ ID NO:2).

FIG. 3 shows PCR growth curves of the nucleic acid test (“DDV) for B.divergens, B. duncani, and B. venatorum on plasmids pEF114 (B.divergens), pEF115 (B. duncani), and pEF116 (B. venatorum) and totalnucleic acids from B. duncani (ATCC PRA 302) (using primers havingnucleic acid sequence of SEQ ID NOs:4 and 6 and a probe having nucleicacid sequence of SEQ ID NO:5).

FIG. 4 shows PCR growth curves of the B. microti oligonucleotide set(“395,” SEQ ID NOs:1-3) and the B. divergens, B. duncani, and B.venatorum oligonucleotide set (“DDV,” SEQ ID NOs:4-6) in a multiplexsetting.

FIG. 5 shows PCR growth curves of the B. microti probe (SEQ ID NO:2) ata spacing of 7 bases between the fluorophore and quencher, in order todetermine optimal spacing.

FIG. 6 shows PCR growth curves of the B. microti probe (SEQ ID NO:2) ata spacing of 10 bases between the fluorophore and quencher, in order todetermine optimal spacing.

FIG. 7 shows PCR growth curves of the B. microti probe (SEQ ID NO:2)evaluated with different dyes (FAM and HEX), in order to determine theoptimal dye.

FIG. 8 shows post-PCR analysis of the B. microti oligonucleotide set(“395,” SEQ ID NOs:1-3), which demonstrates efficient amplification, asevidenced by depletion of oligonucleotides and efficient cleavage of theprobe.

FIG. 9 shows a PCR growth curve for B. microti using the B. microtioligonucleotide set (“395,” SEQ ID NOs:1-3) and the B. divergens, B.duncani, and B. venatorum oligonucleotide set (“DDV,” SEQ ID NOs:4-6) ina multiplex setting in whole blood (treated with cobas PCR media).

FIG. 10 shows PCR growth curves for B. duncani (FIG. 10A), B. divergens(FIG. 10B), and B. venatorum (FIG. 10C) using the B. microtioligonucleotide set (“395,” SEQ ID NOs:1-3) and the B. divergens, B.duncani, and B. venatorum oligonucleotide set (“DDV,” SEQ ID NOs:4-6) ina multiplex setting in whole blood (treated with cobas PCR media).

FIG. 11 shows the overlay of FIGS. 10A, 10B, and 10C in a single figure,showing PCR growth curves for B. duncani, B. divergens, and B.venatorum, using the B. microti oligonucleotide set (“395,” SEQ IDNOs:1-3) and the B. divergens, B. duncani, and B. venatorumoligonucleotide set (“DDV,” SEQ ID NOs:4-6) in a multiplex setting inwhole blood (treated with cobas PCR media).

FIG. 12 shows a PCR growth curve for B. microti using the B. microtioligonucleotide set (“395,” SEQ ID NOs:1-3) and the B. divergens, B.duncani, and B. venatorum oligonucleotide set (“DDVR2,” SEQ ID NOs:4-7,with a mix of equal amounts of reverse primers SEQ ID NOs:6 and 7) in amultiplex setting in whole blood (treated with cobas PCR media).

FIG. 13 shows PCR growth curves for B. duncani (FIG. 13A), B. divergens(FIG. 13B), and B. venatorum (FIG. 13C) using the B. microtioligonucleotide set (“395,” SEQ ID NOs:1-3) and the B. divergens, B.duncani, and B. venatorum oligonucleotide set (“DDVR2,” SEQ ID NOs:4-7,with a mix of equal amounts of reverse primers SEQ ID NOs:6 and 7) in amultiplex setting in whole blood (treated with cobas PCR media).

FIG. 14 shows the overlay of FIGS. 13A, 13B, and 13C in a single figure,showing PCR growth curves for B. duncani, B. divergens, and B.venatorum, using the B. microti oligonucleotide set (“395,” SEQ IDNOs:1-3) and the B. divergens, B. duncani, and B. venatorumoligonucleotide set (“DDV,” SEQ ID NOs:4-6) in a multiplex setting inwhole blood (treated with cobas PCR media)

FIG. 15 shows PCR growth curves for B. duncani, B. divergens, and B.venatorum using a first oligonucleotide set (SEQ ID NOs:1-6) (FIG. 15A)and a second oligonucleotide set (SEQ IQ NOs:1-7, with a mix in equalamounts of reverse primers SEQ ID NOs:6 and 7) (FIG. 15B), in amultiplex setting in whole blood (treated with cobas PCR media).

FIG. 16 shows PCR growth curves for B. duncani, using a firstoligonucleotide set (SEQ ID NOs:1-6) (FIG. 16A) and a secondoligonucleotide set (SEQ IQ NOs:1-7, with a mix in equal amounts ofreverse primers SEQ ID NOs:6 and 7) (FIG. 16B), in a multiplex settingin whole blood (treated with cobas PCR media).

FIG. 17 shows the overlay of FIGS. 16A and 16B in a single figure,showing PCR growth curves for B. duncani, using a first oligonucleotideset (SEQ ID NOs:1-6) (labeled “DDVR”) and a second oligonucleotide set(SEQ IQ NOs:1-7, with a mix in equal amounts of reverse primers SEQ IDNOs:6 and 7) (labeled “DDVR2”), in a multiplex setting in whole blood(treated with cobas PCR media).

DETAILED DESCRIPTION OF THE INVENTION

Diagnosis of Babesia infection by nucleic acid amplification provides amethod for rapidly, accurately, reliably, specifically, and sensitivelydetecting and/or quantitating the Babesia infection. A real-time PCRassay for detecting and/or quantitating Babesia nucleic acids, includingDNA and/or RNA, in a non-biological or biological sample is describedherein. Primers and probes for detecting and/or quantitating Babesia areprovided, as are articles of manufacture or kits containing such primersand probes. The increased specificity and sensitivity of real-time PCRfor detection of Babesia compared to other methods, as well as theimproved features of real-time PCR including sample containment andreal-time detection and quantitating of the amplified product, makefeasible the implementation of this technology for routine diagnosis ofBabesia infections in the clinical laboratory. Additionally, thistechnology may be employed for blood screening as well as for prognosis.This Babesia detection assay may also be multiplexed with other assaysfor the detection of other nucleic acids, e.g., other bacteria and/orviruses, in parallel.

The present disclosure includes oligonucleotide primers and fluorescentlabeled hydrolysis probes that hybridize to the Babesia genome, in orderto specifically identify Babesia using, e.g., TaqMan® amplification anddetection technology.

The disclosed methods may include performing at least one cycling stepthat includes amplifying one or more portions of the nucleic acidmolecule gene target from a sample using one or more pairs of primers.“Babesia primer(s)” as used herein refer to oligonucleotide primers thatspecifically anneal to nucleic acid sequences found in the Babesiagenome, and initiate DNA synthesis therefrom under appropriateconditions producing the respective amplification products. Each of thediscussed Babesia primers anneals to a target such that at least aportion of each amplification product contains nucleic acid sequencecorresponding to the target. The one or more amplification products areproduced provided that one or more nucleic acid is present in thesample, thus the presence of the one or more amplification products isindicative of the presence of Babesia in the sample. The amplificationproduct should contain the nucleic acid sequences that are complementaryto one or more detectable probes for Babesia. “Babesia probe(s)” as usedherein refer to oligonucleotide probes that specifically anneal tonucleic acid sequences found in the Babesia genome. Each cycling stepincludes an amplification step, a hybridization step, and a detectionstep, in which the sample is contacted with the one or more detectableBabesia probes for detection of the presence or absence of Babesia inthe sample.

As used herein, the term “amplifying” refers to the process ofsynthesizing nucleic acid molecules that are complementary to one orboth strands of a template nucleic acid molecule (e.g., nucleic acidmolecules from the Babesia genome). Amplifying a nucleic acid moleculetypically includes denaturing the template nucleic acid, annealingprimers to the template nucleic acid at a temperature that is below themelting temperatures of the primers, and enzymatically elongating fromthe primers to generate an amplification product. Amplificationtypically requires the presence of deoxyribonucleoside triphosphates, aDNA polymerase enzyme (e.g., Platinum® Taq) and an appropriate bufferand/or co-factors for optimal activity of the polymerase enzyme (e.g.,MgCl₂ and/or KCl).

The term “primer” as used herein is known to those skilled in the artand refers to oligomeric compounds, primarily to oligonucleotides butalso to modified oligonucleotides that are able to “prime” DNA synthesisby a template-dependent DNA polymerase, i.e., the 3′-end of the, e.g.,oligonucleotide provides a free 3′-OH group where further “nucleotides”may be attached by a template-dependent DNA polymerase establishing 3′to 5′ phosphodiester linkage whereby deoxynucleoside triphosphates areused and whereby pyrophosphate is released.

The term “hybridizing” refers to the annealing of one or more probes toan amplification product. “Hybridization conditions” typically include atemperature that is below the melting temperature of the probes but thatavoids non-specific hybridization of the probes.

The term “5′ to 3′ nuclease activity” refers to an activity of a nucleicacid polymerase, typically associated with the nucleic acid strandsynthesis, whereby nucleotides are removed from the 5′ end of nucleicacid strand.

The term “thermostable polymerase” refers to a polymerase enzyme that isheat stable, i.e., the enzyme catalyzes the formation of primerextension products complementary to a template and does not irreversiblydenature when subjected to the elevated temperatures for the timenecessary to effect denaturation of double-stranded template nucleicacids. Generally, the synthesis is initiated at the 3′ end of eachprimer and proceeds in the 5′ to 3′ direction along the template strand.Thermostable polymerases have been isolated from Thermus flavus, T.ruber, T. thermophilus, T. aquaticus, T. lacteus, T. rubens, Bacillusstearothermophilus, and Methanothermus fervidus. Nonetheless,polymerases that are not thermostable also can be employed in PCR assaysprovided the enzyme is replenished, if necessary.

The term “complement thereof” refers to nucleic acid that is both thesame length as, and exactly complementary to, a given nucleic acid.

The term “extension” or “elongation” when used with respect to nucleicacids refers to when additional nucleotides (or other analogousmolecules) are incorporated into the nucleic acids. For example, anucleic acid is optionally extended by a nucleotide incorporatingbiocatalyst, such as a polymerase that typically adds nucleotides at the3′ terminal end of a nucleic acid.

The terms “identical” or percent “identity” in the context of two ormore nucleic acid sequences, refer to two or more sequences orsubsequences that are the same or have a specified percentage ofnucleotides that are the same, when compared and aligned for maximumcorrespondence, e.g., as measured using one of the sequence comparisonalgorithms available to persons of skill or by visual inspection.Exemplary algorithms that are suitable for determining percent sequenceidentity and sequence similarity are the BLAST programs, which aredescribed in, e.g., Altschul et al. (1990) “Basic local alignment searchtool” J. Mol. Biol. 215:403-410, Gish et al. (1993) “Identification ofprotein coding regions by database similarity search” Nature Genet.3:266-272, Madden et al. (1996) “Applications of network BLAST server”Meth. Enzymol. 266:131-141, Altschul et al. (1997) “Gapped BLAST andPSI-BLAST: a new generation of protein database search programs” NucleicAcids Res. 25:3389-3402, and Zhang et al. (1997) “PowerBLAST: A newnetwork BLAST application for interactive or automated sequence analysisand annotation” Genome Res. 7:649-656, which are each incorporatedherein by reference.

A “modified nucleotide” in the context of an oligonucleotide refers toan alteration in which at least one nucleotide of the oligonucleotidesequence is replaced by a different nucleotide that provides a desiredproperty to the oligonucleotide. Exemplary modified nucleotides that canbe substituted in the oligonucleotides described herein include, e.g., at-butyl benzyl, a C5-methyl-dC, a C5-ethyl-dC, a C5-methyl-dU, aC5-ethyl-dU, a 2,6-diaminopurine, a C5-propynyl-dC, a C5-propynyl-dU, aC7-propynyl-dA, a C7-propynyl-dG, a C5-propargylamino-dC, aC5-propargylamino-dU, a C7-propargylamino-dA, a C7-propargylamino-dG, a7-deaza-2-deoxyxanthosine, a pyrazolopyrimidine analog, a pseudo-dU, anitro pyrrole, a nitro indole, 2′-O-methyl ribo-U, 2′-O-methyl ribo-C,an N4-ethyl-dC, an N6-methyl-dA, a 5-propynyl dU, a 5-propynyl dC,7-deaza-deoxyguanosine (deaza G (u-deaza)) and the like. Many othermodified nucleotides that can be substituted in the oligonucleotides arereferred to herein or are otherwise known in the art. In certainembodiments, modified nucleotide substitutions modify meltingtemperatures (Tm) of the oligonucleotides relative to the meltingtemperatures of corresponding unmodified oligonucleotides. To furtherillustrate, certain modified nucleotide substitutions can reducenon-specific nucleic acid amplification (e.g., minimize primer dimerformation or the like), increase the yield of an intended targetamplicon, and/or the like in some embodiments. Examples of these typesof nucleic acid modifications are described in, e.g., U.S. Pat. No.6,001,611, which is incorporated herein by reference. Other modifiednucleotide substitutions may alter the stability of the oligonucleotide,or provide other desirable features.

Detection/Quantitation of Babesia Target Nucleic Acid

The present disclosure provides methods to detect Babesia by amplifying,for example, a portion of the Babesia nucleic acid sequence.Specifically, primers and probes to amplify and detect and/or quantitateBabesia nucleic acid molecule targets are provided by the embodiments inthe present disclosure.

For detection and/or quantitation of Babesia, primers and probes toamplify and detect/quantitate the Babesia are provided. Babesia nucleicacids other than those exemplified herein can also be used to detectBabesia in a sample. For example, functional variants can be evaluatedfor specificity and/or sensitivity by those of skill in the art usingroutine methods. Representative functional variants can include, e.g.,one or more deletions, insertions, and/or substitutions in the Babesianucleic acids disclosed herein.

More specifically, embodiments of the oligonucleotides each include anucleic acid with a sequence selected from SEQ ID NOs:1-7, asubstantially identical variant thereof in which the variant has atleast, e.g., 80%, 90%, or 95% sequence identity to one of SEQ IDNOs:1-7, or a complement of SEQ ID NOs:1-7 and the variant.

TABLE 1 Oligonucleotides in Babesia Test SEQ Oligo ID Oligo Name TypeNO: Sequence Modifications 395_21F_TBB Forward 1 ACCTGCTAAATTAGGATCJ: t-Butyl Primer TGGGJ Benzyl-dA 395_56P_HQ10 Sense 2HCTGTTCCAGTQATCGCT H: HEX-Thr Probe TCTTAGAGGGACTTTGCP P: PhosphateQ: BHQ-2 395_123R_TBB Reverse 3 TGTTATTGCCTTACACTT K: t-Butyl PrimerCCTTGK Benzyl-dC DDVF Forward 4 GATGTCCTGGGCTGCJ J: t-Butyl PrimerBenzyl-dA DDVP_HQ8 Anti- 5 HAACTCGATQGAATGCAT H: HEX-Thr SenseCAGTGTAGCGCGP P: Phosphate Probe Q: BHQ-2 DDVR Reverse 6CCCCGTCACGATGCATAC J: t-Butyl Primer TAAJ Benzyl-dA DDVR2 Reverse 7CCCCATCACGATGCATAC J: t-Butyl Primer TAAJ Benzyl-dA

In one embodiment, the above described sets of Babesia primers andprobes are used in order to provide for detection of Babesia in abiological sample suspected of containing Babesia (Table 1). The sets ofprimers and probes may comprise or consist of the primers and probesspecific for the Babesia nucleic acid sequences, comprising orconsisting of the nucleic acid sequences of SEQ ID NOs:1-7. In anotherembodiment, the primers and probes for the Babesia target comprise orconsist of a functionally active variant of any of the primers andprobes of SEQ ID NOs: 1-7.

A functionally active variant of any of the primers and/or probes of SEQID NOs:1-7 may be identified by using the primers and/or probes in thedisclosed methods. A functionally active variant of a primer and/orprobe of any of the SEQ ID NOs:1-7 pertains to a primer and/or probewhich provide a similar or higher specificity and sensitivity in thedescribed method or kit as compared to the respective sequence of SEQ IDNOs:1-7.

The variant may, e.g., vary from the sequence of SEQ ID NOs:1-7 by oneor more nucleotide additions, deletions or substitutions such as one ormore nucleotide additions, deletions or substitutions at the 5′ endand/or the 3′ end of the respective sequence of SEQ ID NOs:1-7. Asdetailed above, a primer and/or probe may be chemically modified, i.e.,a primer and/or probe may comprise a modified nucleotide or anon-nucleotide compound. A probe (or a primer) is then a modifiedoligonucleotide. “Modified nucleotides” (or “nucleotide analogs”) differfrom a natural “nucleotide” by some modification but still consist of abase or base-like compound, a pentofuranosyl sugar or a pentofuranosylsugar-like compound, a phosphate portion or phosphate-like portion, orcombinations thereof. For example, a “label” may be attached to the baseportion of a “nucleotide” whereby a “modified nucleotide” is obtained. Anatural base in a “nucleotide” may also be replaced by, e.g., a7-desazapurine whereby a “modified nucleotide” is obtained as well. Theterms “modified nucleotide” or “nucleotide analog” are usedinterchangeably in the present application. A “modified nucleoside” (or“nucleoside analog”) differs from a natural nucleoside by somemodification in the manner as outlined above for a “modified nucleotide”(or a “nucleotide analog”).

Oligonucleotides including modified oligonucleotides and oligonucleotideanalogs that amplify a nucleic acid molecule encoding the Babesiatarget, e.g., nucleic acids encoding alternative portions of Babesia canbe designed using, for example, a computer program such as OLIGO(Molecular Biology Insights Inc., Cascade, Colo.). Important featureswhen designing oligonucleotides to be used as amplification primersinclude, but are not limited to, an appropriate size amplificationproduct to facilitate detection (e.g., by electrophoresis), similarmelting temperatures for the members of a pair of primers, and thelength of each primer (i.e., the primers need to be long enough toanneal with sequence-specificity and to initiate synthesis but not solong that fidelity is reduced during oligonucleotide synthesis).Typically, oligonucleotide primers are 8 to 50 nucleotides in length(e.g., 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38,40, 42, 44, 46, 48, or 50 nucleotides in length).

In addition to a set of primers, the methods may use one or more probesin order to detect the presence or absence of Babesia. The term “probe”refers to synthetically or biologically produced nucleic acids (DNA orRNA), which by design or selection, contain specific nucleotidesequences that allow them to hybridize under defined predeterminedstringencies specifically (i.e., preferentially) to “target nucleicacids”, in the present case to a Babesia (target) nucleic acid. A“probe” can be referred to as a “detection probe” meaning that itdetects the target nucleic acid.

In some embodiments, the described Babesia probes can be labeled with atleast one fluorescent label. In one embodiment, the Babesia probes canbe labeled with a donor fluorescent moiety, e.g., a fluorescent dye, anda corresponding acceptor moiety, e.g., a quencher. In one embodiment,the probe comprises or consists of a fluorescent moiety and the nucleicacid sequences comprise or consist of SEQ ID NO:3.

Designing oligonucleotides to be used as probes can be performed in amanner similar to the design of primers. Embodiments may use a singleprobe or a pair of probes for detection of the amplification product.Depending on the embodiment, the probe(s) use may comprise at least onelabel and/or at least one quencher moiety. As with the primers, theprobes usually have similar melting temperatures, and the length of eachprobe must be sufficient for sequence-specific hybridization to occurbut not so long that fidelity is reduced during synthesis.Oligonucleotide probes are generally 15 to 40 (e.g., 16, 18, 20, 21, 22,23, 24, or 25) nucleotides in length.

Constructs can include vectors each containing one of Babesia primersand probes nucleic acid molecules (e.g., SEQ ID NOs:1, 2, 3, 4, and 5).Constructs can be used, for example, as control template nucleic acidmolecules. Vectors suitable for use are commercially available and/orproduced by recombinant nucleic acid technology methods routine in theart. Babesia nucleic acid molecules can be obtained, for example, bychemical synthesis, direct cloning from Babesia, or by nucleic acidamplification.

Constructs suitable for use in the methods typically include, inaddition to the Babesia nucleic acid molecules (e.g., a nucleic acidmolecule that contains one or more sequences of SEQ ID NOs:1-5),sequences encoding a selectable marker (e.g., an antibiotic resistancegene) for selecting desired constructs and/or transformants, and anorigin of replication. The choice of vector systems usually depends uponseveral factors, including, but not limited to, the choice of hostcells, replication efficiency, selectability, inducibility, and the easeof recovery.

Constructs containing Babesia nucleic acid molecules can be propagatedin a host cell. As used herein, the term host cell is meant to includeprokaryotes and eukaryotes such as yeast, plant and animal cells.Prokaryotic hosts may include E. coli, Salmonella typhimurium, Serratiamarcescens, and Bacillus subtilis. Eukaryotic hosts include yeasts suchas S. cerevisiae, S. pombe, Pichia pastoris, mammalian cells such as COScells or Chinese hamster ovary (CHO) cells, insect cells, and plantcells such as Arabidopsis thaliana and Nicotiana tabacum. A constructcan be introduced into a host cell using any of the techniques commonlyknown to those of ordinary skill in the art. For example, calciumphosphate precipitation, electroporation, heat shock, lipofection,microinjection, and viral-mediated nucleic acid transfer are commonmethods for introducing nucleic acids into host cells. In addition,naked DNA can be delivered directly to cells (see, e.g., U.S. Pat. Nos.5,580,859 and 5,589,466).

Polymerase Chain Reaction (PCR)

U.S. Pat. Nos. 4,683,202, 4,683,195, 4,800,159, and 4,965,188 discloseconventional PCR techniques. PCR typically employs two oligonucleotideprimers that bind to a selected nucleic acid template (e.g., DNA orRNA). Primers useful in some embodiments include oligonucleotidescapable of acting as points of initiation of nucleic acid synthesiswithin the described Babesia nucleic acid sequences (e.g., SEQ ID NOs:1,2, 4, and 5). A primer can be purified from a restriction digest byconventional methods, or it can be produced synthetically. The primer ispreferably single-stranded for maximum efficiency in amplification, butthe primer can be double-stranded. Double-stranded primers are firstdenatured, i.e., treated to separate the strands. One method ofdenaturing double stranded nucleic acids is by heating.

If the template nucleic acid is double-stranded, it is necessary toseparate the two strands before it can be used as a template in PCR.Strand separation can be accomplished by any suitable denaturing methodincluding physical, chemical or enzymatic means. One method ofseparating the nucleic acid strands involves heating the nucleic aciduntil it is predominately denatured (e.g., greater than 50%, 60%, 70%,80%, 90% or 95% denatured). The heating conditions necessary fordenaturing template nucleic acid will depend, e.g., on the buffer saltconcentration and the length and nucleotide composition of the nucleicacids being denatured, but typically range from about 90° C. to about105° C. for a time depending on features of the reaction such astemperature and the nucleic acid length. Denaturation is typicallyperformed for about 30 sec to 4 min (e.g., 1 min to 2 min 30 sec, or 1.5min).

If the double-stranded template nucleic acid is denatured by heat, thereaction mixture is allowed to cool to a temperature that promotesannealing of each primer to its target sequence. The temperature forannealing is usually from about 35° C. to about 65° C. (e.g., about 40°C. to about 60° C.; about 45° C. to about 50° C.). Annealing times canbe from about 10 sec to about 1 min (e.g., about 20 sec to about 50 sec;about 30 sec to about 40 sec). The reaction mixture is then adjusted toa temperature at which the activity of the polymerase is promoted oroptimized, i.e., a temperature sufficient for extension to occur fromthe annealed primer to generate products complementary to the templatenucleic acid. The temperature should be sufficient to synthesize anextension product from each primer that is annealed to a nucleic acidtemplate, but should not be so high as to denature an extension productfrom its complementary template (e.g., the temperature for extensiongenerally ranges from about 40° C. to about 80° C. (e.g., about 50° C.to about 70° C.; about 60° C.). Extension times can be from about 10 secto about 5 min (e.g., about 30 sec to about 4 min; about 1 min to about3 min; about 1 min 30 sec to about 2 min).

The genome of a retrovirus or RNA virus, is comprised of a ribonucleicacid, i.e., RNA. In such case, the template nucleic acid, RNA, mustfirst be transcribed into complementary DNA (cDNA) via the action of theenzyme reverse transcriptase. Reverse transcriptases use an RNA templateand a short primer complementary to the 3′ end of the RNA to directsynthesis of the first strand cDNA, which can then be used directly as atemplate for polymerase chain reaction.

PCR assays can employ Babesia nucleic acid such as RNA or DNA (cDNA).The template nucleic acid need not be purified; it may be a minorfraction of a complex mixture, such as Babesia nucleic acid contained inhuman cells. Babesia nucleic acid molecules may be extracted from abiological sample by routine techniques such as those described inDiagnostic Molecular Microbiology: Principles and Applications (Persinget al. (eds), 1993, American Society for Microbiology, Washington,D.C.). Nucleic acids can be obtained from any number of sources, such asplasmids, or natural sources including bacteria, yeast, viruses,organelles, or higher organisms such as plants or animals.

The oligonucleotide primers (e.g., SEQ ID NOs:1, 2, 4, and 5) arecombined with PCR reagents under reaction conditions that induce primerextension. For example, chain extension reactions generally include 50mM KCl, 10 mM Tris-HCl (pH 8.3), 15 mM MgCl₂, 0.001% (w/v) gelatin,0.5-1.0 μg denatured template DNA, 50 pmoles of each oligonucleotideprimer, 2.5 U of Taq polymerase, and 10% DMSO). The reactions usuallycontain 150 to 320 μM each of dATP, dCTP, dTTP, dGTP, or one or moreanalogs thereof.

The newly-synthesized strands form a double-stranded molecule that canbe used in the succeeding steps of the reaction. The steps of strandseparation, annealing, and elongation can be repeated as often as neededto produce the desired quantity of amplification products correspondingto the target Babesia nucleic acid molecules. The limiting factors inthe reaction are the amounts of primers, thermostable enzyme, andnucleoside triphosphates present in the reaction. The cycling steps(i.e., denaturation, annealing, and extension) are preferably repeatedat least once. For use in detection, the number of cycling steps willdepend, e.g., on the nature of the sample. If the sample is a complexmixture of nucleic acids, more cycling steps will be required to amplifythe target sequence sufficient for detection. Generally, the cyclingsteps are repeated at least about 20 times, but may be repeated as manyas 40, 60, or even 100 times.

Fluorescence Resonance Energy Transfer (FRET)

FRET technology (see, for example, U.S. Pat. Nos. 4,996,143, 5,565,322,5,849,489, and 6,162,603) is based on a concept that when a donorfluorescent moiety and a corresponding acceptor fluorescent moiety arepositioned within a certain distance of each other, energy transfertakes place between the two fluorescent moieties that can be visualizedor otherwise detected and/or quantitated. The donor typically transfersthe energy to the acceptor when the donor is excited by light radiationwith a suitable wavelength. The acceptor typically re-emits thetransferred energy in the form of light radiation with a differentwavelength. In certain systems, non-fluorescent energy can betransferred between donor and acceptor moieties, by way of biomoleculesthat include substantially non-fluorescent donor moieties (see, forexample, U.S. Pat. No. 7,741,467).

In one example, an oligonucleotide probe can contain a donor fluorescentmoiety or dye (e.g., HEX or FAM) and a corresponding quencher (e.g.,BlackHole Quencher™ (BHQ) (such as BHQ-2)), which may or not befluorescent, and which dissipates the transferred energy in a form otherthan light. When the probe is intact, energy transfer typically occursbetween the donor and acceptor moieties such that fluorescent emissionfrom the donor fluorescent moiety is quenched the acceptor moiety.During an extension step of a polymerase chain reaction, a probe boundto an amplification product is cleaved by the 5′ to 3′ nuclease activityof, e.g., a Taq Polymerase such that the fluorescent emission of thedonor fluorescent moiety is no longer quenched. Exemplary probes forthis purpose are described in, e.g., U.S. Pat. Nos. 5,210,015,5,994,056, and 6,171,785. Commonly used donor-acceptor pairs include theFAM-TAMRA pair. Commonly used quenchers are DABCYL and TAMRA. Commonlyused dark quenchers include BlackHole Quencher™ (BHQ) (such as BHQ2),(Biosearch Technologies, Inc., Novato, Calif.), Iowa Black™, (IntegratedDNA Tech., Inc., Coralville, Iowa), BlackBerry™ Quencher 650 (BBQ-650),(Berry & Assoc., Dexter, Mich.).

In another example, two oligonucleotide probes, each containing afluorescent moiety, can hybridize to an amplification product atparticular positions determined by the complementarity of theoligonucleotide probes to the Babesia target nucleic acid sequence. Uponhybridization of the oligonucleotide probes to the amplification productnucleic acid at the appropriate positions, a FRET signal is generated.Hybridization temperatures can range from about 35° C. to about 65° C.for about 10 sec to about 1 min.

Fluorescent analysis can be carried out using, for example, a photoncounting epifluorescent microscope system (containing the appropriatedichroic mirror and filters for monitoring fluorescent emission at theparticular range), a photon counting photomultiplier system, or afluorimeter. Excitation to initiate energy transfer, or to allow directdetection of a fluorophore, can be carried out with an argon ion laser,a high intensity mercury (Hg) arc lamp, a xenon lamp, a fiber opticlight source, or other high intensity light source appropriatelyfiltered for excitation in the desired range.

As used herein with respect to donor and corresponding acceptor moieties“corresponding” refers to an acceptor fluorescent moiety or a darkquencher having an absorbance spectrum that overlaps the emissionspectrum of the donor fluorescent moiety. The wavelength maximum of theemission spectrum of the acceptor fluorescent moiety should be at least100 nm greater than the wavelength maximum of the excitation spectrum ofthe donor fluorescent moiety. Accordingly, efficient non-radiativeenergy transfer can be produced therebetween.

Fluorescent donor and corresponding acceptor moieties are generallychosen for (a) high efficiency Foerster energy transfer; (b) a largefinal Stokes shift (>100 nm); (c) shift of the emission as far aspossible into the red portion of the visible spectrum (>600 nm); and (d)shift of the emission to a higher wavelength than the Raman waterfluorescent emission produced by excitation at the donor excitationwavelength. For example, a donor fluorescent moiety can be chosen thathas its excitation maximum near a laser line (for example,helium-cadmium 442 nm or Argon 488 nm), a high extinction coefficient, ahigh quantum yield, and a good overlap of its fluorescent emission withthe excitation spectrum of the corresponding acceptor fluorescentmoiety. A corresponding acceptor fluorescent moiety can be chosen thathas a high extinction coefficient, a high quantum yield, a good overlapof its excitation with the emission of the donor fluorescent moiety, andemission in the red part of the visible spectrum (>600 nm).

Representative donor fluorescent moieties that can be used with variousacceptor fluorescent moieties in FRET technology include fluorescein,Lucifer Yellow, B-phycoerythrin, 9-acridineisothiocyanate, LuciferYellow VS, 4-acetamido-4′-isothio-cyanatostilbene-2,2′-disulfonic acid,7-diethylamino-3-(4′-isothiocyanatophenyl)-4-methylcoumarin, succinimdyl1-pyrenebutyrate, and4-acetamido-4′-isothiocyanatostilbene-2,2′-disulfonic acid derivatives.Representative acceptor fluorescent moieties, depending upon the donorfluorescent moiety used, include LC Red 640, LC Red 705, Cy5, Cy5.5,Lissamine rhodamine B sulfonyl chloride, tetramethyl rhodamineisothiocyanate, rhodamine x isothiocyanate, erythrosine isothiocyanate,fluorescein, diethylenetriamine pentaacetate, or other chelates ofLanthanide ions (e.g., Europium, or Terbium). Donor and acceptorfluorescent moieties can be obtained, for example, from Molecular Probes(Junction City, Oreg.) or Sigma Chemical Co. (St. Louis, Mo.).

The donor and acceptor fluorescent moieties can be attached to theappropriate probe oligonucleotide via a linker arm. The length of eachlinker arm is important, as the linker arms will affect the distancebetween the donor and acceptor fluorescent moieties. The length of alinker arm can be the distance in Angstroms (Å) from the nucleotide baseto the fluorescent moiety. In general, a linker arm is from about 10 Åto about 25 Å. The linker arm may be of the kind described in WO84/03285. WO 84/03285 also discloses methods for attaching linker armsto a particular nucleotide base, and also for attaching fluorescentmoieties to a linker arm.

An acceptor fluorescent moiety, such as an LC Red 640, can be combinedwith an oligonucleotide that contains an amino linker (e.g., C6-aminophosphoramidites available from ABI (Foster City, Calif.) or GlenResearch (Sterling, Va.)) to produce, for example, LC Red 640-labeledoligonucleotide. Frequently used linkers to couple a donor fluorescentmoiety such as fluorescein to an oligonucleotide include thiourealinkers (FITC-derived, for example, fluorescein-CPG's from Glen Researchor ChemGene (Ashland, Mass.)), amide-linkers(fluorescein-NHS-ester-derived, such as CX-fluorescein-CPG from BioGenex(San Ramon, Calif.)), or 3′-amino-CPGs that require coupling of afluorescein-NHS-ester after oligonucleotide synthesis.

Detection of Babesia Amplified Product (Amplicon)

The present disclosure provides methods for detecting the presence orabsence of Babesia in a biological or non-biological sample. Methodsprovided avoid problems of sample contamination, false negatives, andfalse positives. The methods include performing at least one cyclingstep that includes amplifying a portion of Babesia target nucleic acidmolecules from a sample using one or more pairs of Babesia primers, anda FRET detecting step. Multiple cycling steps are performed, preferablyin a thermocycler. Methods can be performed using the Babesia primersand probes to detect the presence of Babesia, and the detection ofBabesia indicates the presence of Babesia in the sample.

As described herein, amplification products can be detected usinglabeled hybridization probes that take advantage of FRET technology. OneFRET format utilizes TaqMan® technology to detect the presence orabsence of an amplification product, and hence, the presence or absenceof Babesia. TaqMan® technology utilizes one single-strandedhybridization probe labeled with, e.g., one fluorescent moiety or dye(e.g., HEX or FAM) and one quencher (e.g., BHQ-2), which may or may notbe fluorescent. When a first fluorescent moiety is excited with light ofa suitable wavelength, the absorbed energy is transferred to a secondfluorescent moiety or a dark quencher according to the principles ofFRET. The second moiety is generally a quencher molecule. During theannealing step of the PCR reaction, the labeled hybridization probebinds to the target DNA (i.e., the amplification product) and isdegraded by the 5′ to 3′ nuclease activity of, e.g., the Taq Polymeraseduring the subsequent elongation phase. As a result, the fluorescentmoiety and the quencher moiety become spatially separated from oneanother. As a consequence, upon excitation of the first fluorescentmoiety in the absence of the quencher, the fluorescence emission fromthe first fluorescent moiety can be detected. By way of example, an ABIPRISM® 7700 Sequence Detection System (Applied Biosystems) uses TaqMan®technology, and is suitable for performing the methods described hereinfor detecting the presence or absence of Babesia in the sample.

Molecular beacons in conjunction with FRET can also be used to detectthe presence of an amplification product using the real-time PCRmethods. Molecular beacon technology uses a hybridization probe labeledwith a first fluorescent moiety and a second fluorescent moiety. Thesecond fluorescent moiety is generally a quencher, and the fluorescentlabels are typically located at each end of the probe. Molecular beacontechnology uses a probe oligonucleotide having sequences that permitsecondary structure formation (e.g., a hairpin). As a result ofsecondary structure formation within the probe, both fluorescentmoieties are in spatial proximity when the probe is in solution. Afterhybridization to the target nucleic acids (i.e., amplificationproducts), the secondary structure of the probe is disrupted and thefluorescent moieties become separated from one another such that afterexcitation with light of a suitable wavelength, the emission of thefirst fluorescent moiety can be detected.

Another common format of FRET technology utilizes two hybridizationprobes. Each probe can be labeled with a different fluorescent moietyand are generally designed to hybridize in close proximity to each otherin a target DNA molecule (e.g., an amplification product). A donorfluorescent moiety, for example, fluorescein, is excited at 470 nm bythe light source of the LightCycler® Instrument. During FRET, thefluorescein transfers its energy to an acceptor fluorescent moiety suchas LightCycler®-Red 640 (LC Red 640) or LightCycler®-Red 705 (LC Red705). The acceptor fluorescent moiety then emits light of a longerwavelength, which is detected by the optical detection system of theLightCycler® instrument. Efficient FRET can only take place when thefluorescent moieties are in direct local proximity and when the emissionspectrum of the donor fluorescent moiety overlaps with the absorptionspectrum of the acceptor fluorescent moiety. The intensity of theemitted signal can be correlated with the number of original target DNAmolecules (e.g., the number of Babesia genomes). If amplification ofBabesia target nucleic acid occurs and an amplification product isproduced, the step of hybridizing results in a detectable signal basedupon FRET between the members of the pair of probes.

Generally, the presence of FRET indicates the presence of Babesia in thesample, and the absence of FRET indicates the absence of Babesia in thesample. Inadequate specimen collection, transportation delays,inappropriate transportation conditions, or use of certain collectionswabs (calcium alginate or aluminum shaft) are all conditions that canaffect the success and/or accuracy of a test result, however.

Representative biological samples that can be used in practicing themethods include, but are not limited to whole blood, respiratoryspecimens, urine, fecal specimens, blood specimens, plasma, dermalswabs, nasal swabs, wound swabs, blood cultures, skin, and soft tissueinfections. Collection and storage methods of biological samples areknown to those of skill in the art. Biological samples can be processed(e.g., by nucleic acid extraction methods and/or kits known in the art)to release Babesia nucleic acid or in some cases, the biological samplecan be contacted directly with the PCR reaction components and theappropriate oligonucleotides. In some instances, the biological sampleis whole blood. When whole blood is typically collected, it is oftencollected in vessels containing anticoagulants, such as heparin,citrate, or EDTA, which enables the whole blood to be stored at suitabletemperatures. However, under such conditions, the nucleic acids withinthe whole blood undergo considerable amount of degradation. Therefore,it may be advantageous to collect the blood in a reagent that will lyse,denature, and stabilize whole blood components, including nucleic acids,such as a nucleic acid-stabilizing solution. In such cases, the nucleicacids can be better preserved and stabilized for subsequent isolationand analysis, such as by nucleic acid test, such as PCR. Such nucleicacid-stabilizing solution are well known in the art, including, but notlimited to, cobas PCR media, which contains 4.2 M guanadinium salt(GuHCl) and 50 mM Tris, at a pH of 7.5.

The sample can be collected by any method or device designed toadequately hold and store the sample prior to analysis. Such methods anddevices are well known in the art. In the case that the sample is abiological sample, such as whole blood, the method or device may includea blood collection vessel. Such a blood collection vessel is well knownin the art, and may include, for example, a blood collection tube. Inmany cases, it may be advantageous to use a blood collection tubewherein the blood collection vessel is under pressure in the spaceintended for sample uptake, such as a blood vessel with an evacuatedchamber, such as a vacutainer blood collection tube. Such bloodcollection tubes with an evacuated chamber, such as a vacutainer bloodcollection tube are well known in the art. It may further beadvantageous to collect the blood in a blood collection vessel, with orwithout an evacuated chamber, that contains within it, a solution thatwill lyse, denature, and stabilize whole blood components, includingnucleic acids, such as a nucleic acid-stabilizing solution, such thatthe whole blood being drawn immediately contacts the nucleicacid-stabilizing solution in the blood collection vessel.

Melting curve analysis is an additional step that can be included in acycling profile. Melting curve analysis is based on the fact that DNAmelts at a characteristic temperature called the melting temperature(Tm), which is defined as the temperature at which half of the DNAduplexes have separated into single strands. The melting temperature ofa DNA depends primarily upon its nucleotide composition. Thus, DNAmolecules rich in G and C nucleotides have a higher Tm than those havingan abundance of A and T nucleotides. By detecting the temperature atwhich signal is lost, the melting temperature of probes can bedetermined. Similarly, by detecting the temperature at which signal isgenerated, the annealing temperature of probes can be determined. Themelting temperature(s) of the Babesia probes from the Babesiaamplification products can confirm the presence or absence of Babesia inthe sample.

Within each thermocycler run, control samples can be cycled as well.Positive control samples can amplify target nucleic acid controltemplate (other than described amplification products of target genes)using, for example, control primers and control probes. Positive controlsamples can also amplify, for example, a plasmid construct containingthe target nucleic acid molecules. Such a plasmid control can beamplified internally (e.g., within the sample) or in a separate samplerun side-by-side with the patients' samples using the same primers andprobe as used for detection of the intended target. Such controls areindicators of the success or failure of the amplification,hybridization, and/or FRET reaction. Each thermocycler run can alsoinclude a negative control that, for example, lacks target template DNA.Negative control can measure contamination. This ensures that the systemand reagents would not give rise to a false positive signal. Therefore,control reactions can readily determine, for example, the ability ofprimers to anneal with sequence-specificity and to initiate elongation,as well as the ability of probes to hybridize with sequence-specificityand for FRET to occur.

In an embodiment, the methods include steps to avoid contamination. Forexample, an enzymatic method utilizing uracil-DNA glycosylase isdescribed in U.S. Pat. Nos. 5,035,996, 5,683,896 and 5,945,313 to reduceor eliminate contamination between one thermocycler run and the next.

Conventional PCR methods in conjunction with FRET technology can be usedto practice the methods. In one embodiment, a LightCycler® instrument isused. The following patent applications describe real-time PCR as usedin the LightCycler® technology: WO 97/46707, WO 97/46714, and WO97/46712.

The LightCycler® can be operated using a PC workstation and can utilizea Windows NT operating system. Signals from the samples are obtained asthe machine positions the capillaries sequentially over the opticalunit. The software can display the fluorescence signals in real-timeimmediately after each measurement. Fluorescent acquisition time is10-100 milliseconds (msec). After each cycling step, a quantitativedisplay of fluorescence vs. cycle number can be continually updated forall samples. The data generated can be stored for further analysis.

As an alternative to FRET, an amplification product can be detectedusing a double-stranded DNA binding dye such as a fluorescent DNAbinding dye (e.g., SYBR® Green or SYBR® Gold (Molecular Probes)). Uponinteraction with the double-stranded nucleic acid, such fluorescent DNAbinding dyes emit a fluorescence signal after excitation with light at asuitable wavelength. A double-stranded DNA binding dye such as a nucleicacid intercalating dye also can be used. When double-stranded DNAbinding dyes are used, a melting curve analysis is usually performed forconfirmation of the presence of the amplification product.

One of skill in the art would appreciate that other nucleic acid- orsignal-amplification methods may also be employed. Examples of suchmethods include, without limitation, branched DNA signal amplification,loop-mediated isothermal amplification (LAMP), nucleic acidsequence-based amplification (NASBA), self-sustained sequencereplication (3 SR), strand displacement amplification (SDA), or smartamplification process version 2 (SMAP 2).

It is understood that the embodiments of the present disclosure are notlimited by the configuration of one or more commercially availableinstruments.

Articles of Manufacture/Kits

Embodiments of the present disclosure further provide for articles ofmanufacture or kits to detect Babesia. An article of manufacture caninclude primers and probes used to detect the Babesia gene target,together with suitable packaging materials. Representative primers andprobes for detection of Babesia are capable of hybridizing to Babesiatarget nucleic acid molecules. In addition, the kits may also includesuitably packaged reagents and materials needed for DNA immobilization,hybridization, and detection, such solid supports, buffers, enzymes, andDNA standards. Methods of designing primers and probes are disclosedherein, and representative examples of primers and probes that amplifyand hybridize to Babesia target nucleic acid molecules are provided.

Articles of manufacture can also include one or more fluorescentmoieties for labeling the probes or, alternatively, the probes suppliedwith the kit can be labeled. For example, an article of manufacture mayinclude a donor and/or an acceptor fluorescent moiety for labeling theBabesia probes. Examples of suitable FRET donor fluorescent moieties andcorresponding acceptor fluorescent moieties are provided above.

Articles of manufacture can also contain a package insert or packagelabel having instructions thereon for using the Babesia primers andprobes to detect Babesia in a sample. Articles of manufacture mayadditionally include reagents for carrying out the methods disclosedherein (e.g., buffers, polymerase enzymes, co-factors, or agents toprevent contamination). Such reagents may be specific for one of thecommercially available instruments described herein.

Embodiments of the present disclosure also provide for a set of primersand one or more detectable probes for the detection of Babesia in asample.

Embodiments of the present disclosure will be further described in thefollowing examples, which do not limit the scope of the inventiondescribed in the claims.

EXAMPLES

The following examples and figures are provided to aid the understandingof the subject matter, the true scope of which is set forth in theappended claims. It is understood that modifications can be made in theprocedures set forth without departing from the spirit of the invention.

The test was a fully automated sample preparation (nucleic acidextraction and purification) followed by PCR amplification anddetection. The system used was the Cobas® 6800/8800 System, whichconsisted of a sample supply module, the transfer module, the processingmodule, and the analytic module. Automated data manage was performed bythe Cobas® 6800/8800 System.

Selective amplification of target nucleic acid was achieved by the useof specific forward and reverse primers which were selected from highlyconserved regions of the target nucleic acid. A thermostable DNApolymerase enzyme was used for both reverse-transcription andamplification. The master mix included deoxyuridine triphosphate (dUTP),instead of deoxythimidine triphosphate (dTTP), which is incorporatedinto the newly synthesized DNA (amplified product or amplicon). Anycontaminating amplicons from previous PCR runs were destroyed by theAmpErase enzyme (uracil-N-glycosylase), which was included in the PCRmix, when heated in the first thermal cycling step. Newly formedamplicons were not destroyed, however, since the AmpErase enzyme wasinactivated once exposed to temperatures above 55° C.

The Cobas® Babesia master mix contained detection probes which werespecific for Babesia and control nucleic acids. The specific Babesia andcontrol detection probes were each labeled with one of two uniquefluorescent dyes which act as a reporter. Each probe also had a seconddye which acted as a quencher. The reporter dye is measured at a definedwavelength, thus permitting detection and discrimination of theamplified Babesia target and the control. The fluorescent signal of theintact probes was suppressed by the quencher dye. During the PCRamplification step, hybridization of the probes to the specificsingle-stranded DNA template resulted in cleavage by the 5′ to 3′nuclease activity of the DNA polymerase resulting in separation of thereporter and quencher dyes, and the generation of fluorescent signal.With each PCR cycle, increasing amounts of cleaved probes were generatedand the cumulative signal of the reporter dye was concomitantlyincreased. Because the two specific reporter dyes are measured atdefined wavelengths, simultaneous detection and discrimination of theamplified Babesia target and the control was possible.

The primers and probes for the Babesia test were designed by seedingprimers and probes along the genome in the most conserved regions basedon the alignment. The primers and probes were then combined into assaysand the assays were scored based on the inclusivity and exclusivityin-silico assessment. In addition to genomic conservation, genomiccoverage (which is highly dependent on what sequences are availablepublicly) was also included in the scoring of the assays. The targetedregion of the Babesia genome was the 18S gene for the Babesia species B.microti, B. divergens, B. duncani, and B. venatorum. One set ofoligonucleotides (SEQ ID NOs:1-3) was designed to detect B. microti,which is often referred herein as “395,” and another set ofoligonucleotides (SEQ ID NOs:4-7) was designed to detect B. divergens,B. duncani, and B. venatorum. The set of oligonucleotides designed todetect B. divergens, B. duncani, and B. venatorum included two differentsets of oligonucleotides, as follows: (1) the first set includedoligonucleotides SEQ ID NOs:4-6 (often referred, herein, as “DDVR”); and(2) the second set included oligonucleotides SEQ ID NOs:4-7 (oftenreferred, herein, as “DDVR2”).

Example 1: Amplification and Detection of B. microti by Real-Time PCR

The B. microti nucleic acid test for the Babesia 18S rRNA gene wastested using an oligonucleotide set designed to amplify and detect B.microti (forward primer SEQ ID NO:1, reverse primer SEQ ID NO:3, andprobe SEQ ID NO:2), and using either pEF113 (B. microti strain Gray inpUC19) (FIG. 1) or B. microti genomic DNA (FIG. 2). Reagents usedinclude Cobas® 6800/8800 generic PCR Master Mix, with the profile andconditions for use with the Cobas® 6800/8800, and using TaqMan®amplification and detection technology. The final concentration ofoligonucleotides in the master mix was 0.3 μM for primers and 0.1 μM forprobes. The Cobas® 6800/8800 PCR Profile employed is depicted in Table2, below:

TABLE 2 cobas ® 6800/8800 PCR Profile Target Hold time Step Cycles (°C.) (hh:mm:ss) Ramp Pre-PCR 1 50 00:02:00 4.4 94 00:00:05 4.4 5500:02:00 2.2 60 00:06:00 4.4 65 00:04:00 4.4 1. Meas 5 95 00:00:05 4.455 00:00:30 2.2 2. Meas 45 91 00:00:05 4.4 58 00:00:25 2.2 Post 1 4000:02:00 2.2These studies show that under these conditions, the oligonucleotides(SEQ ID NOs:1-3) were able to amplify and detect B. microti (see, FIGS.1 and 2).

End point dilution series analysis was also performed to assess thelower limit of detection in this system. The B. microti pUC19 plasmid(pEF113) was quantified using ddPCR, diluted, and tested. The results ofthe end-point dilution analysis are shown below in Table 3.

TABLE 3 395 12.5 c/xn 8/8 6.25 c/rxn 8/8 3.13 c/rxn 7/8 1.56 c/rxn 6/80.78 c/rxn 2/8 0.39 c/rxn 0/8 0.20 c/rxn 0/8 0.1 c/rxn 0/8 0.05 c/rxn0/8 0.025 c/rxn 0/8The limit of detection was determined to be 3.32 copies of plasmid DNAper PCR at 95% confidence.

Thus, these results demonstrate that the primers and probes (SEQ IDNOs:1-3) amplify and detect the presence of B. microti efficiently andspecifically in a real-time PCR assay.

Example 2: Amplification and Detection of B. divergens, B. duncani, andB. venatorum by Real-Time PCR

The B. divergens, B. duncani, and B. venatorum (referred here often as“DDV”) nucleic acid test for Babesia 18S rRNA gene was tested using anoligonucleotide set designed to amplify and detect B. divergens, B.duncani, and B. venatorum (forward primer SEQ ID NO:4, reverse primerSEQ ID NO:6, and probe SEQ ID NO:5), and using plasmids pEF114 (B.divergens), pEF115 (B. duncani), and pEF116 (B. venatorum), or totalnucleic acid from B. duncani (ATCC PRA 302) (FIG. 3). Reagents usedinclude Cobas® 6800/8800 generic PCR Master Mix, with the profile andconditions for use with the Cobas® 6800/8800, and using TaqMan®amplification and detection technology. The final concentration ofoligonucleotides in the master mix was 0.3 μM for primers and 0.1 μM forprobes. The Cobas® 6800/8800 PCR Profile employed is depicted in Table2, above. These studies show that under these conditions, theoligonucleotides (SEQ ID NOs:4-6) were able to amplify and detect B.divergens, B. duncani, and B. venatorum (see, FIG. 3).

Thus, these results demonstrate that the primers and probes (SEQ IDNOs:4-6) amplify and detect the presence of B. divergens, B. duncani,and B. venatorum efficiently and specifically in a real-time PCR assay

Example 3: Multiplex Amplification and Detection of B. microti, B.divergens, B. Duncani, and B. venatorum by Real-Time PCR

Because the assay for amplification and detection of B. microti (SEQ IDNOs:1-3; Example 1; and FIGS. 1-2) and the assay for amplification anddetection of B. divergens, B. duncani, and B. venatorum (“DDV,” SEQ IDNOs:4-6; Example 2; and FIG. 3) showed good performance in singleplex,the assays were tested in a multiplex setting, under the same conditionsas described for the singleplex tests.

FIG. 4 shows, as expected, that the B. microti target is amplified anddetected strongly by SEQ ID NOs:1-3, in a multiplex setting, and isamplified and detected weakly by the DDV oligonucleotides (i.e., SEQ IDNOs:4-6). Similarly, FIG. 4 also shows that B. divergens, B. duncani,and B. venatorum are not amplified and detected by SEQ ID NOs:1-3, butare strongly amplified and detected by the DDV oligonucleotides (i.e.,SEQ ID NOs:4-6).

Example 4: Optimization of Spacings Between Fluorophore and Quencher ofProbe

The B. microti probe (i.e., SEQ ID NO:2) was evaluated with differentspacings between the fluorophore and quencher to determine the optimalspacing (see, FIG. 5). The assays were tested under the same conditionsas described previously, in Examples 1-3. Although the probe waseffective at a spacing of seven and 10 bases in between fluorophore andquencher, the 10-spaced probe exhibited similar endpoint RFI and betterinclusivity (see, FIGS. 5 and 6).

Thus, the Babesia probes are efficient and specific within a wide rangeof spacing between the fluorophore and quencher, including between 7-10bases.

Example 5: Optimization of Probe Dyes

The B. microti probe (i.e., SEQ ID NO:2) was evaluated with differentfluorescent moieties or fluorescent dyes, FAM and HEX. Assays weretested under the same conditions as described previously, in Examples1-4. Although the probe was effective with either FAM or HEX fluorescentmoieties/dyes, the HEX-labeled probe demonstrated a lower baseline,leading to greatly increased signal (see, FIG. 7).

Thus, the Babesia probes are efficient and specific with a number ofdifferent types of fluorescent moieties/dyes, including FAM and HEX.

Example 6: Post-PCR Analysis

A post-PCR analysis was performed with the oligonucleotides detecting B.microti (SEQ ID NOs:1-3). This post-PCR analysis was performed in orderto ensure efficient amplification as evidenced by depletion ofoligonucleotides and efficient cleavage of the probe. As can be seen inFIG. 8, the B. microti oligonucleotides (SEQ ID NOs:1-3) demonstratesdepletion of oligonucleotides and probe cleavage.

Thus, the Babesia oligonucleotides ensure efficient amplification, asevidenced by depletion of oligonucleotides and efficient cleavage of theprobe.

Example 7: Multiplex Amplification and Detection of B. microti, B.divergens, B. Duncani, and B. venatorum by Real-Time PCR in Whole Blood

The oligonucleotides for amplification and detection of B. microti, B.divergens, B. duncani, and B. venatorum were tested in whole blood.Briefly, secondary standard was made by lysing Babesia culture in cobasPCM media (CPM). Cobas PCR media is a pre-analytic reagent that lyses,denatures, and stabilizes whole blood components, including nucleicacids. Cobas PCR media contains guanidinium salt (here, GuHCl at 4.2 M)and Tris (here, 50 mM), at a pH of 7.5. Four separate standards for fourdifferent Babesia species (B. microti, B. divergens, B. duncani, and B.venatorum) were generated in this manner. The secondary standard wasdiluted to intermediate levels in cobas PCR media, then spiked into awhole blood:cobas PCR media mixture. The whole blood:cobas PCR mediamixture is 1 part whole blood, and 7 parts cobas PCR media. The finalconcentrations of each standard were as shown below in Table 4.

TABLE 4 Standard Concentrations Babesia Strain Concentration (iRBC/ml)B. microti 0.375 B. divergens 5.700 B. duncani 4.088 B. venatorum 500

The standard-spiked whole blood was subjected to oligonucleotides foramplification and detection of B. microti, B. divergens, B. duncani, andB. venatorum (SEQ ID NOs:1-3 and 4-6), under conditions as describedpreviously (Examples 1-6). Results are shown in FIG. 9-11. FIG. 9reveals that the oligonucleotide set of SEQ ID NOs:1-3 to detect B.microti in combination with the oligonucleotide set of SEQ ID NOs:4-6 todetect B. divergens, B. duncani, and B. venatorum (in a multiplexsetting) were able to specifically and efficiently amplify and detect B.microti in whole blood. FIGS. 10-11 reveal that the oligonucleotide setof SEQ ID NOs:1-3 to detect B. microti in combination with theoligonucleotide set of SEQ ID NOs:4-6 to detect B. divergens, B.duncani, and B. venatorum (in a multiplex setting) were able tospecifically and efficiently amplify and detect B. divergens (FIGS. 10Aand 11), B. duncani (FIGS. 10B and 11), and B. venatorum (FIGS. 10C and11) in whole blood. Thus, these results demonstrate that theoligonucleotide set of SEQ ID NOs:1-6 specifically and efficientlyamplify and detect B. microti, B. divergens, B. duncani, and B.venatorum in whole blood. These results also demonstrate that cobas PCRmedia that lyses, denatures, and stabilizes whole blood components,including nucleic acids

Example 8: Multiplex Amplification and Detection of B. microti, B.divergens, B. Duncani, and B. venatorum by Real-Time PCR in Whole Bloodwith Pair of Reverse Primers for B. divergens, B. duncani, and B.venatorum

Further studies were conducted to demonstrate multiplex amplificationand detection of B. microti, B. divergens, B. duncani, and B. venatorumby real-time PCR in whole blood, as above in Example 7, but with use ofa pair of reverse primers for amplification and detection of B.divergens, B. duncani, and B. venatorum. That is, a new oligonucleotideset for the amplification and detection of B. divergens, B. duncani, andB. venatorum was tested. In particular, a new reverse primer, SEQ IDNO:7 was used in concert with reverse primer SEQ ID NO:6. The twodifferent reverse primers (SEQ ID NOs:6 and 7) were then used incombination with forward primer SEQ ID NO:4 and probe SEQ ID NO:5,designed to amplify and detect B. divergens, B. duncani, and B.venatorum. The oligonucleotide set of SEQ ID NOs:4-7 designed to detectB. divergens, B. duncani, and B. venatorum was then combined with theoligonucleotide set of SEQ ID NOs:1-3 designed to detect B. microti toinvestigate of the combined oligonucleotide set of SEQ ID NOs:1-7 couldamplify and detect B. microti, B. divergens, B. duncani, and B.venatorum in whole blood. The conditions were identical to as describedfor the previous whole blood studies described previously. The finalconcentrations of each standard were as shown below in Table 5.

TABLE 5 Standard Concentrations Babesia Strain Concentration (iRBC/ml)B. microti 0.375 B. divergens 5.700 B. duncani 4.088 B. venatorum 12.5

Results are shown in FIG. 12-14. FIG. 12 reveals that theoligonucleotide set of SEQ ID NOs:1-3 to detect B. microti incombination with the oligonucleotide set of SEQ ID NOs:4-7 to detect B.divergens, B. duncani, and B. venatorum (in a multiplex setting) wereable to specifically and efficiently amplify and detect B. microti inwhole blood. FIGS. 13-14 reveal that the oligonucleotide set of SEQ IDNOs:1-3 to detect B. microti in combination with the oligonucleotide setof SEQ ID NOs:4-7 to detect B. divergens, B. duncani, and B. venatorum(in a multiplex setting) were able to specifically and efficientlyamplify and detect B. divergens (FIGS. 13A and 14), B. duncani (FIGS.13B and 14), and B. venatorum (FIGS. 13C and 14) in whole blood.

These experiments were then reproduced to compare the oligonucleotideset SEQ ID NOs:1-6 versus the oligonucleotide set SEQ ID NOs:1-7 intheir abilities to detect and amplify B. microti (data not shown), B.divergens, B. duncani, and B. venatorum in whole blood. These resultsare shown in FIG. 15. The oligonucleotide set of SEQ ID NOs:1-7 employedtwo reverse primers, SEQ ID NOs:6 and 7, in equal amounts. FIG. 15 showsthe curves for B. divergens, B. duncani, and B. venatorum using theoligonucleotide set of SEQ ID NOs:1-6 (FIG. 15A) versus theoligonucleotide set of SEQ ID NOs:1-7 (FIG. 15B). In particular, the PCRcurves for B. duncani were analyzed (FIGS. 16-17). These data revealthat the oligonucleotide set SEQ ID NO:1-7 (FIG. 16B) exhibited improvedamplification of B. duncani as compared to the oligonucleotide set ofSEQ ID NO:6 (FIG. 16A). That is, while the oligonucleotide set of SEQ IDNO:1-6 (which included a single reverse primer, SEQ ID NO:6, to amplifyB. divergens, B. duncani, and B. venatorum) is able to successfullyamplify and detect B. divergens, B. duncani, and B. venatorum, theoligonucleotide set of SEQ ID NOs:1-7 (which included two reverseprimers, SEQ ID NO:6 and 7 in equal amounts, to amplify B. divergens, B.duncani, and B. venatorum), the pair of reverse primers (SEQ ID NOs:6and 7) exhibited improved amplification of B. duncani (FIG. 17).

Thus, these results demonstrate that the oligonucleotide set of SEQ IDNOs:1-6 specifically and efficiently amplify and detect B. microti, B.divergens, B. duncani, and B. venatorum in whole blood. These resultsalso demonstrate that cobas PCR media that lyses, denatures, andstabilizes whole blood components, including nucleic acids.

While the foregoing invention has been described in some detail forpurposes of clarity and understanding, it will be clear to one skilledin the art from a reading of this disclosure that various changes inform and detail can be made without departing from the true scope of theinvention. For example, all the techniques and apparatus described abovecan be used in various combinations. All publications, patents, patentapplications, and/or other documents cited in this application areincorporated by reference in their entirety for all purposes to the sameextent as if each individual publication, patent, patent application,and/or other document were individually indicated to be incorporated byreference for all purposes.

1. A method of detecting the Babesia species of (i) B. microti, and/or(ii) B. divergens, B. duncani, and/or B. venatorum in a sample, themethod comprising: (a) performing an amplification step comprisingcontacting the sample with one or more set of primers to produce anamplification product, if a target nucleic acid of (i) B. microti,and/or (ii) B. divergens, B. duncani, and/or B. venatorum is present inthe sample; (b) performing a hybridization step comprising contactingthe amplification product, if the target nucleic acid of (i) B. microti,and/or (ii) B. divergens, B. duncani, and/or B. venatorum is present inthe sample, with one or more probes; and (c) detecting the presence orabsence of the amplification product, wherein the presence of theamplification product is indicative of the presence of (i) B. microti,and/or (ii) B. divergens, B. duncani, and/or B. venatorum in the sample,and wherein the absence of the amplification product is indicative ofthe absence of (i) B. microti, and/or (ii) B. divergens, B. duncani,and/or B. venatorum in the sample; and wherein the one or more set ofprimers and the one or more probes comprise: (i) a set of primers foramplification of B. microti comprising: a primer comprising the nucleicacid sequence of SEQ ID NO:1, or a complement thereof, and a primercomprising the nucleic acid sequence of SEQ ID NO:3, or a complementthereof; and a probe for hybridizing to the amplification product of B.microti comprising the nucleic acid sequence of SEQ ID NO:2, or acomplement thereof; and/or (2) a set of primers for amplification of B.divergens, B. duncani, and/or B. venatorum comprising: a primercomprising the nucleic acid sequence of SEQ ID NO:4, or a complementthereof, and one or more primers compromising the nucleic acidsequence(s) of SEQ ID NO:6 or SEQ ID NO:7 or a combination of SEQ IDNOs:6 and 7, or a complement thereof; and a probe for hybridizing to theamplification product of B. divergens, B. duncani, and/or B. venatorumcomprising the nucleic acid sequence of SEQ ID NO:5, or a complementthereof. 2-13. (canceled)
 14. The method of claim 1, wherein the sampleis a biological sample.
 15. The method of claim 14, wherein thebiological sample is whole blood, respiratory specimens, urine, fecalspecimens, blood specimens, plasma, dermal swabs, nasal swabs, woundswabs, blood cultures, skin, or soft tissue infections.
 16. The methodof claim 15, wherein the biological sample is whole blood.
 17. Themethod of claim 1, wherein the hybridization step comprises contactingthe amplification product with the one or more probes, wherein the oneor more probes is labeled with a donor fluorescent moiety and acorresponding acceptor moiety; and the detecting step comprisesdetecting the presence or absence of fluorescent resonance energytransfer (FRET) between the donor fluorescent moiety and the acceptormoiety of the one or more probes, wherein the presence or absence offluorescence is indicative of the presence or absence of Babesia in thesample.
 18. The method of claim 17, wherein the donor fluorescent moietyis HEX or FAM.
 19. The method of claim 18, wherein the donor fluorescentmoiety is HEX.
 20. The method of claim 17, wherein the acceptor moietyis a quencher.
 21. The method of claim 20, wherein the quencher isBlackHole Quencher™-2 (BHQ-2).
 22. The method of claim 17, wherein thedonor fluorescent moiety and the acceptor moiety are within 5 to 20nucleotides of each other.
 23. The method of claim 22, wherein the donorfluorescent moiety and the acceptor moiety are within 7 to 10nucleotides of each other.
 24. The method of claim 23, wherein the donorfluorescent moiety and the acceptor moiety are within 8 nucleotides ofeach other.
 25. The method of claim 23, wherein the donor fluorescentmoiety and the acceptor moiety are within 10 nucleotides of each other.26. A method of detecting Babesia of (i) B. microti, and/or (ii) B.divergens, B. duncani, and/or B. venatorum in a sample, wherein thesample is whole blood, the method comprising: (a) performing anamplification step comprising contacting the sample with one or more setof primers to produce an amplification product, if a target nucleic acidof (i) B. microti, and/or (ii) B. divergens, B. duncani, and/or B.venatorum is present in the whole blood sample; (b) performing ahybridization step comprising contacting the amplification product, ifthe target nucleic acid of (i) B. microti, and/or (ii) B. divergens, B.duncani, and/or B. venatorum is present in the sample, with one or moreprobes; and (c) detecting the presence or absence of the amplificationproduct, wherein the presence of the amplification product is indicativeof the presence of (i) B. microti, and/or (ii) B. divergens, B. duncani,and/or B. venatorum in the sample, and wherein the absence of theamplification product is indicative of the absence of (i) B. microti,and/or (ii) B. divergens, B. duncani, and/or B. venatorum in the sample;and wherein the one or more set of primers and the one or more probescomprise: (1) a set of primers for amplification of B. microticomprising: a primer comprising the nucleic acid sequence of SEQ IDNO:1, or a complement thereof; and a primer compromising the nucleicacid sequence of SEQ ID NO:3, or a complement thereof; and a probe forhybridizing to the amplification product of B. microti comprising thenucleic acid sequence of SEQ ID NO:2, or a complement thereof; and/or(2) a set of primers for amplification of B. divergens, B. duncani,and/or B. venatorum comprising: a primer comprising the nucleic acidsequence of SEQ ID NO:4, or a complement thereof, and one or moreprimers compromising the nucleic acid sequence(s) of SEQ ID NO:6 or SEQID NO:7 or a combination of SEQ ID NOs:6 and 7, or a complement thereof;and a probe for hybridizing to the amplification product of B.divergens, B. duncani, and/or B. venatorum comprising the nucleic acidsequence of SEQ ID NO:5, or a complement thereof. 27-64. (canceled) 65.A method of detecting the Babesia species of B. microti in a sample,wherein the sample is whole blood, the method comprising: (a) performingan amplification step comprising contacting the sample with one or moreset of primers to produce an amplification product, if a target nucleicacid of B. microti is present in the whole blood sample; (b) performinga hybridization step comprising contacting the amplification product, ifthe target nucleic acid of B. microti is present in the sample, with oneor more probes; and (c) detecting the presence or absence of theamplification product, wherein the presence of the amplification productis indicative of the presence of B. microti in the sample, and whereinthe absence of the amplification product is indicative of the absence ofB. microti in the sample; and wherein the one or more set of primers andthe one or more probes comprise: (1) a set of primers for amplificationof B. microti comprising: (i) a primer comprising the nucleic acidsequence of SEQ ID NO:1, or a complement thereof, and (ii) a primercompromising the nucleic acid sequence of SEQ ID NO:3, or a complementthereof; and (2) a probe for hybridizing to the amplification product ofB. microti comprising the nucleic acid sequence of SEQ ID NO:2, or acomplement thereof.
 66. A method of detecting the Babesia species of B.divergens, B. duncani, and/or B. venatorum in a sample, wherein thesample is whole blood, the method comprising: (a) performing anamplification step comprising contacting the sample with one or more setof primers to produce an amplification product, if a target nucleic acidof B. divergens, B. duncani, and/or B. venatorum is present in the wholeblood sample; (b) performing a hybridization step comprising contactingthe amplification product, if the target nucleic acid of B. divergens,B. duncani, and/or B. venatorum is present in the sample, with one ormore probes; and (c) detecting the presence or absence of theamplification product, wherein the presence of the amplification productis indicative of the presence of B. divergens, B. duncani, and/or B.venatorum in the sample, and wherein the absence of the amplificationproduct is indicative of the absence of B. divergens, B. duncani, and/orB. venatorum in the sample; and wherein the one or more set of primersand the one or more probes comprise: (1) a set of primers foramplification of B. divergens, B. duncani, and/or B. venatorumcomprising, (i) a primer comprising the nucleic acid sequence of SEQ IDNO:4, or a complement thereof and (ii) one or more primers compromisingthe nucleic acid sequence(s) of SEQ ID NO:6 or SEQ ID NO:7 or acombination of SEQ ID NOs:6 and 7, or a complement thereof; and (2) aprobe for hybridizing to the amplification product of B. divergens, B.duncani, and/or B. venatorum comprising the nucleic acid sequence of SEQID NO:5, or a complement thereof.
 67. The method of claim 65 or 66,wherein the sample is a biological sample.
 68. The method of claim 67,wherein the biological sample is whole blood, respiratory specimens,urine, fecal specimens, blood specimens, plasma, dermal swabs, nasalswabs, wound swabs, blood cultures, skin, or soft tissue infections. 69.The method of claim 68, wherein the biological sample is whole blood.70. The method of claim 65 or 66, wherein the hybridization stepcomprises contacting the amplification product with the one or moreprobes, wherein the one or more probes is labeled with a donorfluorescent moiety and a corresponding acceptor moiety; and thedetecting step comprises detecting the presence or absence offluorescent resonance energy transfer (FRET) between the donorfluorescent moiety and the acceptor moiety of the one or more probes,wherein the presence or absence of fluorescence is indicative of thepresence or absence of Babesia in the sample.
 71. The method of claim70, wherein the donor fluorescent moiety is HEX or FAM.
 72. The methodof claim 71, wherein the donor fluorescent moiety is HEX.
 73. The methodof claim 70, wherein the acceptor moiety is a quencher.
 74. The methodof claim 73, wherein the quencher is BlackHole Quencher™-2 (BHQ-2). 75.The method of claim 70, wherein the donor fluorescent moiety and theacceptor moiety are within 5 to 20 nucleotides of each other.
 76. Themethod of claim 75, wherein the donor fluorescent moiety and theacceptor moiety are within 7 to 10 nucleotides of each other.
 77. Themethod of claim 76, wherein the donor fluorescent moiety and theacceptor moiety are within 8 nucleotides of each other.
 78. The methodof claim 76, wherein the donor fluorescent moiety and the acceptormoiety are within 10 nucleotides of each other.